Glaucoma is a major blinding disease. We propose a new study on optineurin, a gene identified in 2002 that is associated principally with low tension or normal tension glaucoma. In particular, we will examine the cellular processing of optineurin in neuronal cells. Proper processing of cellular proteins is of vital importance. In eukaryotic cells, the ubiquitin-proteasome system (UPS) and autophagy pathway are two major routes for protein clearance. Optineurin protein has ubiquitin binding domain. Preliminary studies performed also suggest that the endogenous optineurin is ubiquitinated and processed through UPS in retinal ganglion RGC5 cells. We have also noted that overexpressed wild type or mutant optineurin may be associated with autophagy and cell death. The central hypotheses are: i) in normal homeostatic situation, the turnover of endogenous optineurin involves UPS;ii) when optineurin is overexpressed or mutated, proteasome activity is impaired and autophagy comes into play, similar to that observed in neurodegenerative Alzheimer's and Parkinson's diseases;and iii) overexpressed and mutated optineurin leads to retinal ganglion cell (RGC) death. Three specific aims are proposed. Both in vitro culture and in vivo animal systems will be used. In Specific Aim 1, the turnover of endogenous optineurin in RGC5 cells and its ubiquitination will be studied. Specific proteasome and autophagy inhibitors/stimulator will be used in experiments including pulse chase to determine whether the degradation rate of optineurin would be affected. Molecular chaperones associated with endogenous optineurin will be identified. In Specific Aim 2, the involvement of UPS and autophagy in RGC5 cells after transfection to overexpress wild type and E50K optineurin and in inducible PC12 or RGC5 cell lines will be investigated. Experiments will be designed to determine whether proteasome activity is impaired with optineurin overexpression or mutation, whether autophagy is induced, and whether cell death is a consequence. The rescuing studies will be performed in Specific Aim 3. We will also deliver optineurin gene using adeno-associated viruses into the retinal ganglion cells of rat eyes to examine whether impairment of UPS, induction of autophagy and cell death take place in vivo, whether the overexpressed optineurin can be efficiently cleared, and whether cell death is reduced by rescuing treatments. We believe that the results obtained through the proposed studies will be valuable, providing not only basic information regarding biology of optineurin but also suggesting a neurodegenerative disease paradigm for glaucoma. Moreover, the rescuing efforts will potentially have high translational significance. PUBLIC HEALTH RELEVANCE Glaucoma is a major blinding disease. We propose a new study on optineurin, a gene associated with normal tension glaucoma. In particular, we will examine the processing of optineurin protein in neuronal cells. Results obtained from the proposed studies will provide insights into the basic biology of optineurin and help illustrate how glaucoma is developed. In addition, the new information may lead to novel therapeutic modalities.